This invention relates to treatment of viral infections using organic compounds which interact with T-cell enzymes.
One of the classic markers of full-blown AIDS resulting from long-term infection with HIV-1 is a severe depletion of CD4+ T-cells, which are a key component of the immune system. Attempts have been made to increase the CD4+ counts of AIDS patients, and some of these efforts, notably treatment with protease inhibitors, have met with considerable success. Other approaches, e.g., stimulation of the immune response by vaccination with viral peptides, have been less successful. The reasons for CD4+ depletion in AIDS, and resistance of CD4+ cells to stimulation by some therapies, are not fully understood.
We have discovered that the activation state of human T-cells can be affected by compounds which interact with a cytoplasmic post-prolyl dipeptidase activity which has similarities to, but is distinct from, the membrane-bound T-cell serine protease CD26. The compounds useful in the invention are inhibitors of this activity, which is, in naturally-occurring T-cells in healthy individuals, involved in protection of T-cells from apoptosis, or programmed cell death. Thus, in high concentrations, the inhibitors hasten the death of T-cells, by inhibiting the protective enzyme. We have discovered, surprisingly, that at low concentrations the inhibitors exhibit a paradoxical effect: they are potent stimulators of T-cell activity in HIV-infected individuals. The concentrations of inhibitor which induce this T-cell stimulatory response are very low (on the order of 10xe2x88x928-10xe2x88x9212M), and therefore the inhibitors can be used with minimal side effects, even if, in larger doses, the inhibitors would be toxic.
Our hypothesis is that the resistance to full activation observed in T-cells of HIV-infected individuals involves a blocking of the cytoplasmic enzymatic activity discussed above. We believe that this blocking of activation, involving this cytoplasmic activity, prevents differentiation of T-cells of HIV-infected individuals into effector cells, eventually leading to T-cell death.
Thus, the invention features a method for stimulating proliferation of T-cells of a human patient suffering from a disease state characterized by the inability of the patients"" T-cells to respond normally to T-cell proliferation-inducing stimuli; the method involves contacting the T-cells with an organic compound at a concentration below 10xe2x88x928M, wherein the compound is characterized in that it binds to the post-prolyl cleaving dipeptidase activity present in the cytoplasm of human T-cells, e.g., CD4+ cells or Jurkat cells.
Treatment according to the invention can be in vitro or in vivo. In in vivo therapy, the enzyme-interacting compound of the invention is administered such that the blood concentration in the patient (e.g., an HIV-infected patient) is below 10xe2x88x9211. The compounds can also be used in vitro at low concentrations to stimulate proliferation of non-infected, beneficial T-cells, such as CD4+ cells and CTL""s. In this embodiment, PBMC are isolated from a patient and incubated with a concentration of lower than 10xe2x88x928M of the compound, to bring about proliferation of T-cells, which are then reinfused into the patient.
We believe that administration of low concentrations of the inhibitors of the invention may have an allosteric effect such that the T-cell cytoplasmic enzyme, which is a multimeric (i.e., multiple subunit) enzyme, exhibits an increased affinity of the enzyme for its natural substrate or ligands, allowing the previously blocked T-cell to proceed to full activation, and hence survival, proliferation, and interleukin-2 production. Stimulation of the T-cell immune response in HIV-infected patients according to the invention yields increased numbers of immune effector cells, which can fight both HIV itself, and other opportunistic pathogens.
Treatment according to the invention has the advantages of specificity and low toxicity, not just because of the low concentrations of inhibitor which can be used, but also because, in T-cells of patients not infected with a virus such as HIV, the inhibitors have no discernable effect. Furthermore, treatment according to the invention advantageously does not necessarily require in vitro manipulation of the T-cells from HIV-infected patients. Furthermore, no immunization is required, and treatment will be effective even where HIV proteins have mutated because the therapy targets a cellular enzyme. The fact that, in T-cells treated according to the invention in vitro, no increase in the level of the HIV protein p24 is observed, probably indicates that the T-cells which are infected with HIV are not stimulated by the low dose inhibitor treatment of the invention.
The invention also permits immunization of HIV-infected patients with, e.g., HIV peptides. Under normal circumstances, such patients cannot be vaccinated because of the defect in the T-cell stimulation pathway. Use of inhibitors in low doses as adjuvants can render T-cells responsive to vaccination with HIV antigens, in particular peptides.
Treatment of HIV-infected patients with low doses of inhibitors according to the invention can also enhance the activity of other AIDS drugs, in particular protease inhibitors. We have found that treatment according to the invention generally fails to bring about an increase in CD4+ count in patients whose CD4+ count is already very low, i.e., below about 400. In such patients, the CD4+ count can be increased to above this level using known protease inhibitors, and the newly generated CD4+ T-cells resulting from such treatment are particularly susceptible to the stimulatory effects of treatment according to the invention, leading to an optimal combination AIDS therapy. Preferably, the drugs are administered orally.
The low dose administration of inhibitors of the invention can also be used to produce an adjuvant effect in HIV-negative individuals, who are to be immunized with peptides or other viral antigens; this mode of vaccination can be used for prophylaxis for HIV, as well as any other viral pathogen. Ordinarily, meaningful cytolytic T-lymphocyte (xe2x80x9cCTLxe2x80x9d) responses, both in vitro and in vivo, have been difficult to achieve with peptide immunization. The invention should make it possible to produce significant CTL responses to viral peptides, e.g., peptides from influenza, HIV, human papilloma virus, and herpes peptides. This adjuvant effect can also be used to stimulate CTL responses to peptide antigens from other pathogens as well, e.g., pathogenic bacteria such as toxigenic E. coli, and protozoan pathogens such as the pathogens which are the causative agents of malaria and amoebic dysentery. The compounds, when used as adjuvants, are preferably administered orally.
The invention provides a new and highly advantageous method of potentiating the immune response in both HIV infected and uninfected patients, in methods employing extremely low concentrations of inhibitors which, at these concentrations, exhibit a paradoxical effect (i.e., they act as stimulatory rather than inhibitory molecules, as they would at higher concentrations). The very low concentrations employed according to the invention allows treatment with minimal side reactions and toxicity. The specificity of the treatment of the invention also avoids such adverse effects, which are seen, for example, in treatment with immune stimulatory compounds such as interleukin-2.
Other features and advantages of the invention will be apparent from the following detailed description thereof, and from the claims.